Ribonuclease III (RNase III) is an endoribonuclease that cleaves double stranded RNA. The enzyme is expressed in many organisms and is highly conserved. I. S. Mian, Nucleic Acids Res., 1997, 25, 3187-95. All RNase III species cloned to date contain an RNase III signature sequence and vary in size from 25 to 50 kDa. Multiple functions have been ascribed to RNase III. In both E. coli and S. cerevisiae, RNase III has been reported to be involved in the processing of pre-ribosomal RNA (pre-rRNA). Elela et al., Cell, 1996, 85, 115-24. RNase III has also been reported to be involved in the processing of small molecular weight nuclear RNAs (snRNAs) and small molecular weight nucleolar RNAs (snoRNAs) in S. cerevisiae. Chanfreau et al., Genes Dev. 1996, 11, 2741-51; Qu et al., Mol. Cell. Biol. 1996, 19, 1144-58. In E. coli, RNase III has also been reported to be involved in the degradation of some mRNA species. D. Court, in Control of messenger RNA stability, 1993, Academic Press, Inc, pp. 71-116.
A human double strand RNase (dsRNase) activity has been described. Wu et al., J.Biol. Chem., 1998, 273, 2532-2542; Crooke, U.S. Pat. No. 5,898,031; U.S. Pat. No. 6,107,094. By the rational design and testing of chemically modified antisense oligonucleotides that contained oligoribonucleotide stretches of varying length, a dsRNase activity was demonstrated in human T24 bladder carcinoma cells which produced 5′-phosphate and 3′-hydroxyl termini upon cleavage of the complementary cellular RNA target. This pattern of cleavage products is a feature of E. coli RNase III. The cleavage activity in human cells required the formation of a dsRNA region in the oligonucleotide. This human dsRNase activity is believed to be useful as an alternative terminating mechanism to RNase H for antisense therapeutics. Because it relies on “RNA-like” oligonucleotides, which generally have higher potency than the “DNA-like” oligonucleotides required for RNase H activity, it may prove an attractive alternative to RNase H-based antisense approaches.
RNA interference (RNAi) is a form of sequence-specific, post-transcriptional gene silencing in animals and plants, elicited by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. Elbashir et al., Nature, 2001, 411, 494-498. dsRNA triggers the specific degradation of homologous RNAs, only within the region of homology. The dsRNA is processed to 21- to 23-nucleotide fragments, sometimes called short interfering RNAs (siRNAs) which are believed to be the guide fragments for sequence-specific mRNA degradation. The processing of longer dsRNA to these short siRNA fragments is believed to be accomplished by RNase III. Elbashir et al., ibid., Elbashir et al., Genes and Devel., 2001, 15, 188-200. Thus it is believed that the human RNase III of the present invention may be useful in further understanding and exploiting the gene silencing mechanism, particularly in human cells.
Despite the substantial information about members of the RNase III family and the cloning of genes encoding proteins with RNase III activity from a number of lower organisms (E. coli, yeast and others), no human RNase III has previously been cloned. This has hampered efforts to understand the structure of the enzyme(s), its distribution and the functions it may serve. The present application describes the cloning and characterization of a cDNA that expresses a human RNase III. Cloning and sequencing of the cDNA encoding human RNase III allowed characterization of this nucleic acid as well as of the location and function of the RNase III protein itself.